Doug Auld, Ph.D. Novartis Institutes for BioMedical Research

iPSCs Are an Essential Tool for Modeling How Causal Genetic Variants Impact Cellular Function in Disease

This paper comes from several major stem cell centers, and describes the automation and workflow for scaling induced pluripotent stem cells (iPSCs). The automated system is detailed, along with online videos of the process, and the overall workflow is presented (see figure). To show the robustness of the system, 640 skin tissue samples were used to provide fibroblast cultures. Twenty of these were karyotyped before starting, and 95% showed a normal diploid karyotype. Sendai virus was initially used to provide the iPSCs, but residual expression of the Sendai virus and variable efficiencies were noted, so automated delivery of mRNA for the transcription factors was employed. With this method, out of 523 reprogramming attempts, 221 were successful (judged by TRA-1-60-positive colonies). The production of iPSCs is performed in 96-well plates, and further analysis of pluripotent markers showed that the system could provide low passage iPSC of high purity in this format. Next, the system expands the iPSCs and harvests these into barcoded cryotubes. Karyotyping supports that 89% of these had a normal karyotype (n=38). Examination of embryoid bodies (EBs) produced on the automation showed lower variation in gene expression profiles compared to manually derived EBs. The main source for the difference in variability between automated and manual methods appears to be technical differences in manual selection of iPSC colonies. In the automated method, iPSCs were purified by negatively selecting against poorly reprogrammed cells using immunomagnetic beads for separation resulting in a 26-fold enrichment of reprogrammed cells. These were arrayed in 96-well plates, and the wells were imaged to determine polyclonal populations with similar growth profiles. Flow cytometry showed that 80% of the selected colonies showed pluripotent markers. Of note, cell passage differences did not greatly influence the cell characteristics. However, the age of the donor samples was found to hinder the production of iPSCs, with older age samples showing poorer production.

Furthermore, the growth rate and confluency during the start of reprogramming were found to influence successful reprogramming greatly. Overall, the automated system described in this paper can provide 100s of iPSC samples per month with reduced reagent cost. This paper should be a valuable resource for those attempting to scale iPSC production.

* Abstract from Nat Methods 2015;12:885–892

Induced pluripotent stem cells (iPSCs) are an essential tool for modeling how causal genetic variants impact cellular function in disease, as well as an emerging source of tissue for regenerative medicine. The preparation of somatic cells, their reprogramming and the subsequent verification of iPSCs pluripotency are laborious, manual processes limiting the scale and reproducibility of this technology. Here we describe a modular, robotic platform for iPSCs reprogramming enabling automated, high-throughput conversion of skin biopsies into iPSCs and differentiated cells with minimal manual intervention. We demonstrate that automated reprogramming and the pooled selection of polyclonal pluripotent cells results in high-quality, stable iPSCs. These lines display less line-to-line variation than either manually produced lines or lines produced through automation followed by single-colony subcloning. The robotic platform we describe will enable the application of iPSCs to population-scale biomedical problems including the study of complex genetic diseases and the development of personalized medicines.

Doug Auld, Ph.D., is affiliated with the Novartis Institutes for BioMedical Research.

ASSAY & Drug Development Technologies, published by Mary Ann Liebert, Inc., offers a unique combination of original research and reports on the techniques and tools being used in cutting-edge drug development. The journal includes a "Literature Search and Review" column that identifies published papers of note and discusses their importance. GEN presents here one article that was analyzed in the "Literature Search and Review" column, a paper published in Nature Methods titled "Automated, high-throughput derivation, characterization and differentiation of induced pluripotent stem cells." Authors of the paper are Paull D, Sevilla A, Zhou H, Hahn AK, Kim H, Napolitano C, Tsankov A, Shang L, Krumholz K, Jagadeesan P, Woodard CM, Sun B, Vilboux T, Zimmer M, Forero E, Moroziewicz DN, Martinez H, Malicdan MC, Weiss KA, Vensand LB, Dusenberry CR, Polus H, Sy KT, Kahler DJ, Gahl WA, Solomon SL, Chang S, Meissner A, Eggan K, Noggle SA.

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